Potential Use of Antifreeze DNA Aptamers for the Cryopreservation of Human Erythrocytes

Abstract

This article summarizes proof of concept experiments which clearly demonstrated the ability of DNA aptamers selected against a molecular mimic of early ice crystal nuclei to protect the integrity of human erythrocytes following a slow freeze-thaw cycle. Following 10 cycles of selection and DNA amplification of rare candidate DNA aptamers against a copper-organic ligand complex which holds water molecules in a conformation resembling early ice crystal nuclei, the aptamers were tested for their ability to preserve erythrocyte morphology by phase-contrast microscopy versus controls without aptamers and with the original randomized aptamer DNA library template following slow freezing and storage overnight at -20 °C with slow thawing at 25 °C. Those experiments revealed that a minimum of 32 μg/ml of the final selected aptamer pool of DNA molecules was required to completely protect nearly 100% of the erythrocytes. By contrast, the treatment groups without the aptamers or with the randomized aptamer template DNA at 32 μg/ml produced only fragmented erythrocytes and cellular debris (no intact cells), thus indicating that specifically selected DNA conformations were required to bind and limit the size of forming ice crystals to cryoprotect the erythrocytes.